The goal of this proposal is the continued development of DNA sequencing by charge reduction electrospray ionization. The research presented here focuses on the development of new techniques for the generation ionization and sampling of gas-phase biomolecular ions. The combination of these techniques holds the potential to produce a l0,000 fold increase in sensitivity over conventional electrospray. Single droplets will be generated by a piezoelectric dispenser and held in an electrodynamic trap where they will be desolvated by a flow of bath gas to produce analyte ions. The analyte ions will leave the trap along a defined trajectory to enter a charge reduction chamber incorporating aerodynamic lensing. The aerodynamic lens system will replace the standard nozzle-skimmer and collisional cooling systems commonly employed in ESI-TOF. The fact that the ions will travel along a single defined trajectory, rather than along a multitude of trajectories as in conventional electrospray will permit time collection efficiency of the ions into time mass spectrometer to be greatly increased. In principle, the efficiency with which ions are introduced mass spectrometer may approach unity representing an approximately 10,000 fold increase over conventional electrospray. Additionally techniques to increase the ionization efficiency of DNA through gas phase reactions and charge tagging will be researched. The focus of this proposal will be the implementation of the basic concepts outlined above. To this end we will utilize aim existing ESI-TOF mass spectrometer (Perseptive Biosystems Mariner an orthogonal-TOF design) in our laboratory. The use of this existing instrument will eliminate the need to design and build an entirely new mass spectrometer from scratch while permitting dramatic improvements in nmass spectrometric performance.